SUMMARY

The muscles that power swimming in the blue crab, Callinectes
sapidus, grow hypertrophically, such that in juvenile crabs the cell
diameters are <60 μm, whereas fibers of the adult crabs often exceed 600μ
m. Thus, as these animals grow, their muscle fibers greatly exceed the
surface area to volume ratio and intracellular diffusion distance limits of
most cells. Previous studies have shown that arginine phosphate (AP) recovery
in the anaerobic (light) fibers, which demonstrate a fiber size dependence on
anaerobic processes following contraction, is too slow to be restricted by
intracellular metabolite diffusive flux, in spite of the fiber's large size.
By contrast, the aerobic (dark) fibers have evolved an intricate network of
intracellular subdivisions that maintain an effectively small `metabolic
diameter' throughout development. In the present study, we examined the impact
of intracellular metabolite diffusive flux on the rate of post-contractile AP
resynthesis in the dark muscle, which has a much higher aerobic capacity than
the light muscle. AP recovery was measured for 60 min in adults and 15 min in
juveniles following burst contractile activity in dark fibers, and a
mathematical reaction-diffusion model was used to test whether the observed
aerobic rates of AP resynthesis were fast enough to be limited by
intracellular metabolite diffusion. Despite the short diffusion distances and
high mitochondrial density, the AP recovery rates were relatively slow and we
found no evidence of diffusion limitation. However, during simulation of
steady-state contraction, which is an activity more typical of the dark
fibers, there were substantial intracellular metabolite gradients, indicative
of diffusion limitation. This suggests that high ATP turnover rates may lead
to diffusion limitation in muscle even when diffusion distances are short, as
in the subdivided dark fibers.

Introduction

The muscles that power swimming in the blue crab, Callinectes
sapidus, grow by increasing the diameter of individual fibers
(hypertrophy), rather than by increasing fiber number (hyperplasia), and
during post-metamorphic development fiber diameters increase from <60 μm
in juveniles to >600 μm in adults
(Boyle et al., 2003). This
contrasts with muscle fibers of most organisms, which generally have a size
range of 10-100 μm. Presumably, fiber size is governed by the fundamental
need to carry out aerobic metabolic processes, which rely on oxygen flux
across cell membranes (Kim et al.,
1998), and ATP diffusive flux from mitochondria to sites of ATP
demand (Mainwood and Rakusan,
1982). Thus, a likely functional consequence of excessive cell
size is a reduced capacity for oxidative metabolism
(Boyle et al., 2003;
Johnson et al., 2004;
Kinsey et al., 2005).

The swimming muscles of C. sapidus are composed of three distinct
types of fibers: light fibers that power anaerobic burst swimming, dark fibers
that power aerobically fueled endurance swimming, and a small number of fibers
intermediate to the light and dark fibers
(Tse et al., 1983). The
anaerobic light fibers rely on endogenous fuels such as arginine phosphate
(AP) and glycogen during contraction, not oxygen influx, so contractile
function should not be impacted by an increase in fiber size. However,
aerobically driven processes, such as post-contractile recovery, may be
limited in the largest light fibers because low cell surface area to volume
ratios may constrain oxygen flux into the cell and intracellular diffusion
distances may become excessive (Kinsey and
Moerland, 2002; Boyle et al.,
2003; Kinsey et al.,
2005). These cellular level limitations may therefore have
behavioral costs by extending the recovery time required between successive
bursts of high velocity swimming needed for predator escape.

Whereas the dark fibers reach the same large dimensions as the light
fibers, their aerobic contractile function should favor small size throughout
development. To accommodate the conflicting demands for hypertrophic growth
and small fiber size, dark fibers have evolved small mitochondria-rich
subdivisions (Tse et al.,
1983) that increase in number and maintain a constant size during
development, as well as promote intra-fiber perfusion to facilitate
O2 delivery to the subdivisions
(Johnson et al., 2004). Thus,
blue crab dark fibers are unusual in having metabolic functional units (fiber
subdivisions) that retain small dimensions throughout development, whereas
their contractile functional units (fibers) appear to grow hypertrophically to
extreme proportions.

Anaerobic light fibers are therefore characterized by large cell size and
low ATP demand, whereas dark fibers remain effectively small (via
subdivisions) throughout development, but have the capacity for much higher
rates of ATP turnover. We have previously hypothesized that anaerobic
glycogenolysis is recruited following burst contractions in large anaerobic
fibers to accelerate certain key phases of recovery that would otherwise be
slowed by size-related limitations to the rate of aerobic ATP synthesis
(Kinsey and Moerland, 2002;
Boyle et al., 2003;
Johnson et al., 2004;
Kinsey et al., 2005). This
hypothesis is supported by observations that the rate of post-contractile AP
resynthesis, which is potentially increased by anaerobic metabolism, is
size-independent in blue crab anaerobic fibers
(Kinsey et al., 2005).
Further, significant post-contractile glycogen depletion
(Boyle et al., 2003) and
increased post-contractile lactate accumulation
(Johnson et al., 2004), both
anticipated consequences of anaerobic glycogenolysis, were found in the
anaerobic fibers from adult animals, but not juveniles. It is unlikely,
however, that this strategy is implemented in the large dark fibers because
intracellular subdivisions maintain the small effective diameter necessary to
permit aerobic metabolism during recovery. Thus, it is reasonable to expect
that differences in the rate of post-contractile AP recovery in dark fibers
from adult and juvenile crabs are not related to fiber size, but result from
`normal' metabolic scaling with body mass (Schmidt-Neilson, 1984).
Furthermore, if anaerobic glycogenolysis is not being exploited in the large
aerobic fibers, post-contractile glycogen depletion and lactate accumulation
should be minimal and size independent.

Johnson et al. (Johnson et al.,
2004), however, reported significant post-contractile lactate
accumulation in the highly subdivided dark fibers of adult crabs (although the
levels were significantly lower than seen in light fibers). The authors
reasoned that this was a likely consequence of close proximity of the dark
muscle to the much larger mass of lactate-producing light fibers, as well as
net diffusive flux into the dark fibers from the lactate-laden hemolymph, but
not the result of post-contractile anaerobic glycogenolysis occurring within
the dark fibers. The absence of size-dependent glycogen depletion in dark
fibers would be consistent with the conclusions of Johnson et al.
(Johnson et al., 2004).

These previous observations provide strong evidence for fiber size effects
in blue crab light muscle fibers, which are probably mediated through
excessive intracellular diffusive distances and/or low cell surface area to
volume ratios. The dark fibers have the aforementioned structural
modifications that appear to offset the constraints of large fiber size, but
these fibers also have high rates of ATP turnover that make them more
susceptible to diffusion limitation. The present study examined the fiber size
dependence of post-contractile recovery in the dark muscle fibers of juvenile
and adult blue crabs. The objectives were to (1) measure the rate of AP
recovery, (2) apply a mathematical reaction-diffusion model to determine
whether the rate of AP recovery is limited by intracellular metabolite
diffusive flux, and (3) measure post-contractile glycogen depletion. We
hypothesized that (1) differences in the rate of post-contractile AP recovery
in dark fibers are principally the result of differences in mass-specific
aerobic capacity due to metabolic scaling (Schmidt-Neilson, 1984); (2)
intracellular metabolite diffusive flux does not limit the rate of AP
recovery; and (3) there is no size-dependent post-contractile depletion of
glycogen in dark aerobic fibers because anaerobic metabolism is not recruited
during recovery in the large fibers of the adults.

Materials and methods

Animals

Juvenile blue crabs (Callinectes sapidus Rathbun) were collected
by sweep netting in the basin of the Cape Fear River Estuary, NC, USA. Adult
crabs were obtained from baited crab traps set in Masonboro Sound, NC, USA or
purchased from local fisherman (Wilmington, NC, USA). Crabs were maintained in
full-strength filtered seawater (35‰ salinity, 21°C) in aerated,
recirculating aquariums. They were fed bait shrimp three times weekly and kept
on a 12 h:12 h light:dark cycle. All animals were held under these conditions
for at least 72 h before experimentation. Animals were sexed, and their
carapace width and body mass were measured prior to use
(Table 1). Only animals in the
intermolt stage were used as determined by the rigidity of the carapace, the
presence of the membranous layer of the carapace, and the absence of a soft
cuticle layer developing beneath the existing exoskeleton
(Roer and Dillaman, 1984).

Exercise protocol

Crabs were induced to undergo a burst swimming response as described
previously (Boyle et al., 2003;
Johnson et al., 2004;
Kinsey et al., 2005). Crabs
were held suspended in the air by a clamp in a manner that allowed free motion
of the swimming legs and small wire electrodes were placed in two small holes
drilled into the mesobranchial region of the dorsal carapace. A Grass
Instruments SD9 physiological stimulator (Astro Med, Inc., West Warwick, RI,
USA) was used to deliver a small voltage (80 Hz, 200 ms duration, 10 V
cm-1 between electrodes) to the thoracic ring ganglia, which
elicited a burst swimming response in the fifth periopods for several seconds
following the stimulation. A single pulse was administered every 20-30 s until
the animal was no longer capable of a burst response, which was evident when
it responded by moving its legs at a notably slower rate. During exercise,
animals were exposed to the air for a period of only 3-4 min, which is
sufficiently short to avoid compromised gill oxygen transport due to changing
scaphognathite activity, lamellar clumping or lactate accumulation
(deFur et al., 1988).
Immediately following exercise, animals were returned to aerated full-strength
seawater and allowed to recover. Animals assayed for AP were sampled at 0, 15,
30 or 60 min (adults) and 0, 5, 10 or 15 min (juveniles) post-contraction,
whereas animals assayed for glycogen were sampled at 0, 30, 60, 120 or 240 min
post-contraction.

Metabolite measurement

At the end of the recovery period, crabs were rapidly cut in half along
their sagittal plane in order to minimize the spontaneous burst contraction of
the swimming legs that typically occurs as they are killed. The dorsal
carapace, reproductive and digestive organs were removed and the basal cavity
that houses the muscles of the fifth periopod was exposed. The dark levator
muscle was rapidly isolated by cutting away the surrounding muscle and
freeze-clamped using tongs cooled in liquid nitrogen while still intact within
the animal. The time elapsed from death to freeze clamping the muscle was
60-90 s. After tissue extraction, samples being analyzed for glycogen were
stored at -80°C until further evaluation. Samples assayed for AP were
immediately homogenized in a 6- to 35-fold dilution of chilled 7% perchloric
acid with 1 mmol l-1 EDTA using a Fisher Powergen 125 homogenizer,
and then centrifuged at 16 000 g for 30 min at 4°C. The
supernatant pH was neutralized with 3 mol l-1 potassium bicarbonate
in 50 mmol l-1 Pipes, stored on ice for 10 min, and centrifuged at
16 000 g for 15 min at 4°C. The supernatant was
immediately analyzed by 31P nuclear magnetic resonance (NMR)
spectroscopy. NMR spectra were collected at 162 MHz on a Bruker 400 DMX
spectrometer (Bruker Instruments, Billerica, MA, USA) to determine relative
concentrations of AP, inorganic phosphate (Pi), and ATP. Spectra
were collected using a 90° excitation pulse and a relaxation delay of 12
s, which ensures that the phosphorus nuclei were fully relaxed and peak
integrals for the metabolites were proportional to their relative
concentrations. Forty-eight scans were acquired for a total acquisition time
of 10 min. The area under each peak was integrated using Xwin-NMR software to
yield relative concentrations of each metabolite.

Previously frozen tissue samples were analyzed for glycogen based on the
method of Keppler and Decker (Keppler and
Decker, 1974). Samples were homogenized in a 5- to 31-fold
dilution of 3.6% perchloric acid then divided into two pools: a blank aliquot
for measuring free glucose, and a sample aliquot for measuring total glucose
content (free glucose+glycogen). The total glucose sample aliquot was
neutralized with 1 mol l-1 potassium bicarbonate and incubated at
40°C for 2 h in an amyloglucosidase solution (14 units ml-1 in
0.2 mol l-1 acetate buffer, pH 4.8) while undergoing constant
shaking. After incubation was complete, the reaction was stopped by the
addition of 3.6% perchloric acid and both the glucose blank and the total
glucose sample were centrifuged at 16 000 g for 15 min. Before
being assayed supernatants from both pools were neutralized with 1 mol
l-1 KHCO3. Both aliquots were then added to a solution
containing 1 mol l-1 ATP, 0.9 mmol l-1 β-NADP, 15.6
units glucose 6-phosphate dehydrogenase, 0.3 mol l-1
triethanolamine hydrochloride, and 4.05 mmol l-1 MgSO4
at pH 7.5. The reaction was started by the addition of 15.8 units of
hexokinase. The amount of glucose in each pool is proportional to the increase
in NADPH, which was measured spectrophotometrically at a wavelength of 340 nm.
Subtraction of the free glucose in the blank from glucose hydrolyzed from
glycogen in the total glucose sample aliquot yielded glycogen content in units
of μmol glucosyl g-1 of tissue.

Mathematical modeling

The reaction-diffusion model used in the present study was as described in
Kinsey et al. (Kinsey et al.,
2005), with parameters adjusted to comply with blue crab dark
levator fibers. In brief, the model calculated the diffusion and reaction of
ATP, ADP, AP, arginine (Arg) and Pi in a one-dimensional system
that extended from the surface of a mitochondrion to a distance (λ/2)
equal to half of the mean free spacing between clusters of mitochondria, which
were assumed to be distributed at the periphery of each subdivision
(Fig. 1). Four kinetic
expressions were used to determine reaction rates, and these expressions were
either boundary reactions (i.e. the production of ATP at the mitochondrial
membrane), or bulk reactions (those reactions that occur throughout the
cytoplasm). Michaelis-Menten expressions were used for the mitochondrial
boundary reaction (ADP+Pi→ATP) with a rate dependent on the
ADP concentration, a myosin ATPase bulk reaction (ATP→ADP+Pi)
that is only active during contraction, and a basal ATPase bulk reaction that
is always active. In addition, a complete kinetic expression for arginine
kinase (AK) was included in the bulk phase
(Smith and Morrison, 1969).
Diffusion coefficients for radial motion (perpendicular to the fiber long
axis; D ⊥) incorporated the time-dependence of
diffusion found in skeletal muscle (Kinsey
et al., 1999; Kinsey and
Moerland, 2002). Temporally and spatially dependent concentration
profiles of metabolites were calculated using molar-species continuity
equations for all five metabolites (Bird et
al., 1960).

Schematic of the reaction-diffusion mathematical model. Metabolite
concentrations during a contraction-recovery cycle in dark levator fibers were
modeled over the length λ/2, which represents half of the distance
between mitochondrial clusters.

Simulations of a burst contraction-recovery cycle were generated using the
finite element analysis software, FEMLAB (Comsol, Inc., Burlington, MA, USA).
The myosin ATPase was activated at 10 Hz for several seconds, while the basal
ATPase was active throughout the entire contraction-recovery cycle. Model
input parameters are detailed in Table
2. The resting metabolite concentrations for crustacean aerobic
locomotor fibers were obtained from data gathered during this study and
calculations using the AK equilibrium constant
(Teague and Dobson, 1999).
Metabolite data, expressed as μmol g-1 muscle tissue, were
converted to mmol l-1 by assuming that intracellular water
accounted for 68% of the wet mass in blue crab dark levator muscle
(Milligan et al., 1989).
Resting arginine concentrations were set at a reasonable, but arbitrary value
(Beis and Newsholme, 1975). The
D ⊥ values for each metabolite were based both on
direct measurements from crustacean anaerobic fibers and calculations from the
relationship of molecular mass and D ⊥ in these
fibers (Kinsey and Moerland,
2002). Intracellular diffusion distances were estimated according
to Johnson et al. (Johnson et al.,
2004), who found a mean subdivision diameter of 35.6 μm and a
primarily subsarcolemmal distribution of mitochondria in the subdivisions of
both small and large fibers in the dark muscle. A Km for
the mitochondrial reaction (Km,mito) for ADP of 50 μmol
l-1 was used, which is within the range for slow-twitch skeletal
muscle (Kushmerick et al.,
1992). The basal ATPase maximal velocity
(Vm,bas) and Km
(Km,bas) for ATP were estimated so as to maintain constant
resting concentrations over time in an inactive fiber and to promote a return
to the initial steady state following contraction, and these values are
similar to basal ATPase rates estimated for skeletal muscle
(Vicini and Kushmerick, 2000).
AK dissociation constants were obtained from Smith and Morrison
(Smith and Morrison, 1969),
the maximal velocity for the reverse reaction (Vm,AK,rev)
was taken from measurements in blue crab dark levator muscle
(Holt and Kinsey, 2002), and
the maximal velocity for the forward reaction (Vm,AK,for)
was calculated from the AK Haldane relationship from Smith and Morrison
(Smith and Morrison, 1969)
using an equilibrium constant for AK of 40
(Teague and Dobson, 1999). The
myosin ATPase maximal velocity (Vm,myo) and
Km (Km,myo) for ATP were the same as
used for aerobic muscle by Hubley et al.
(Hubley et al., 1997).

Although the model generated temporally and spatially resolved
concentrations of metabolites, our experimental measurements yielded values
that were spatially averaged across the fiber. In order to compare the model
results with the experimental data, some of the model data was mathematically
volume averaged to remove the spatial dependence in concentration while
retaining the temporal variation. (Note that since the model is
one-dimensional, this averaging process required integration only over that
one dimension.) For model simulations that were volume averaged, the duration
of myosin ATPase activation was adjusted so that the decrease in AP
concentration ([AP]) was comparable to that in the observed data and the
values for the maximal velocity of the mitochondrial reaction
(Vm,mito) values were adjusted so that the AP recovery
curve, predicted by the model, approximated the measured recovery rate. This
approach facilitated the analysis of diffusion limitation of the rate of AP
recovery. Since the dark levator muscle is active during sustained aerobic
swimming, steady-state contractions, in which myosin was continuously active,
were also simulated, and Vm,mito and
Vm,myo were adjusted to model different rates of ATP
turnover. These latter simulations were arbitrarily modeled in the small
fibers, because of the similarity in the model parameters between the large
and small fibers and because the higher rate of oxidative phosphorylation in
the small fibers make them more likely to be influenced by intracellular
metabolite diffusion.

Analysis

Measurements of AP during recovery were not collected at the same time
points for both small and large animals, so a t-test was used to
compare the fractional recovery at 15 min post-exercise, a recovery time point
that was shared between size classes. For other metabolite data, a one-way
ANOVA was used to test for the main effects of recovery time. Where
significant differences were detected, Tukey's HSD tests were used to compare
post-contractile recovery time points to the resting value. All metabolite
data are presented as means ± s.e.m. with a significance accepted at
P<0.05.

Results

Metabolite recovery

Size classes were defined based on the relationship between animal mass and
light levator fiber size determined by Boyle et al.
(Boyle et al., 2003)
(Table 1), where the mean fiber
size in the small and large animals was approximately 150 μm and 600 μm,
respectively. In these experiments, as in previous studies
(Boyle et al., 2003;
Johnson et al., 2004;
Kinsey et al., 2005), the crab
stimulation procedure elicited a burst escape response that was qualitatively
similar for both size classes. The frequency of leg beats during burst
swimming was higher in the juveniles, but the duration of the movement was
greater in the adults, and both size classes required approximately the same
number of stimulations to reach fatigue. Additionally, glycogen depletion
(Boyle et al., 2003), lactate
accumulation (Johnson et al.,
2004) and AP depletion (Kinsey
et al., 2005) (see below) during exercise were identical in muscle
fibers from juvenile and adult crabs, indicating a uniform metabolic response
to stimulated exercise.

During a burst exercise-recovery cycle there is a reciprocal change in AP
and Pi that results from the stoichiometric coupling of cellular
ATPases and the AK reaction. Contraction results in a rapid depletion of AP,
and corresponding increase in Pi, which is followed by a slow
recovery to pre-contractile levels. This pattern is demonstrated in examples
of the 31P-NMR spectra collected from perchloric acid extracts of
dark muscle (Fig. 2).
Table 3 shows the absolute
concentrations of metabolites collected at rest, and the time course of
relative changes in the AP and Pi content during a
contraction-recovery cycle are shown in
Fig. 3. In the large fibers,
total recovery takes about 60 min, while the small fibers completely recover
in about 15 min. A comparison of the percentage AP recovery at 15 min
post-exercise revealed a significant difference (t-test,
P<0.05) between size classes (mean values for small and large
fibers were 100.94±10.14% and 46.27±8.9% of the resting value
after 15 min of recovery, respectively). During the course of a
contraction-recovery cycle, ATP content and the sum of AP, Pi, and ATP
remained constant in both large and small fibers, as expected
(Fig. 3).

Absolute resting values of AP, Pi, ATP and glycogen in small
and large dark levator fibers

The absolute amount of glycogen at rest can be found in
Table 3. The relative glycogen
values during a contraction-recovery cycle in both size classes are
illustrated in Fig. 4. The
values at each time point have been normalized to the mean resting values to
allow a direct comparison of post-contractile glycogen changes in small and
large animals. In the large size class there was no significant depletion for
up to 4 h after contraction, although there was a transient, non-significant,
decrease in glycogen immediately after exercise (F=0.7611, d.f.=5,
P=0.5818). In the small size class, however, there was a significant
depletion of glycogen during recovery (F=6.38, d.f.=5,
P=0.0001). Glycogen values at 60, 120 and 240 min after exercise were
significantly lower than values in animals at rest or immediately
post-exercise.

Reaction-diffusion analysis of contraction and recovery

The modeled rate of mitochondrial ATP production was adjusted to
approximate our measured AP recovery curve, thereby allowing us to test
whether the aerobic synthesis of AP was limited by metabolite diffusion in the
dark levator fibers. The model results were volume-averaged to allow a direct
comparison of the observed and simulated recovery rates.
Fig. 5 shows that the model and
measured data for both size classes are in close agreement, thus permitting us
to draw conclusions about diffusion limitation in the aerobic fibers. The
spatially and temporally resolved concentrations of high-energy phosphate
molecules during a contraction-recovery cycle are also presented in
Fig. 5. As expected, neither
fiber exhibited intracellular concentration gradients, indicating that
diffusive flux is fast relative to the rate of ATP turnover.

Representative 31P-NMR spectra collected from perchloric acid
extracts of large dark levator muscle fibers that demonstrate the changes in
relative concentrations of AP and Pi during a contraction-recovery
cycle. Spectra were collected from crabs at rest, and after 0, 30 and 60 min
of recovery from burst exercise. The same pattern of recovery was observed in
the small dark fibers, except that complete AP resynthesis occurred in only 15
min. Chemical shifts are in units of parts per million.

Although the contraction-recovery protocol used here is experimentally
tractable, the primary function of the dark fibers is to power sustained,
steady-state contraction. However, this is a condition we cannot replicate
experimentally, so we used the model to simulate steady-state contraction.
Fig. 6 shows the effect of
increments in the rate of the mitochondrial boundary reaction and the myosin
ATPase (i.e. turnover) on [AP] and [ATP] during steady-state contraction. The
initial simulation (Fig. 6A,B),
in which AP is depleted by roughly 50% during contraction, is a realistic
depiction of a steady-state contraction in skeletal muscle
(Meyer, 1988) and both the
boundary reaction and myosin ATPase values used in this simulation are
reasonable estimates (Vicini and
Kushmerick, 2000). There are obvious AP gradients across the cell,
indicating that at the high rate of ATP turnover characteristic of
steady-state contraction, diffusion is limiting. As the mitochondrial boundary
reaction and myosin ATPase activity are increased to simulate higher-intensity
swimming, a much greater AP depletion is observed, which reduces the cell's
ability to buffer [ATP], leading to substantial intracellular ATP gradients
(Fig. 6C-F). Although aerobic
dark fibers may not be limited by diffusive flux during recovery from burst
contraction, it appears that they are limited during sustained steady-state
exercise.

Discussion

The principal findings of the present study were that (1) the rate of AP
recovery following exercise in the aerobic dark fibers was dependent on body
mass, with differences somewhat greater than that expected from normal
metabolic scaling; (2) intracellular diffusive flux does not appear to limit
aerobic AP recovery after burst contraction, but it does appear to limit
aerobic flux during steady-state contraction; and (3) there was no significant
glycogen depletion during post-contractile recovery in the large fibers, which
is consistent with our hypothesis that intracellular subdivisions alleviate
the need for anaerobic contributions during recovery.

Relative changes in AP (A) and Pi (B) content and absolute
changes in ATP (C) and AP+Pi+ATP (D) content in small (open
symbols) and large (filled symbols) dark levator fibers during a
contraction-recovery cycle. In A and B, values at each time point have been
normalized to the mean immediately after contraction to allow direct
comparison of the recovery rate between the size classes (absolute resting
values are in Table 3). Note
how quickly AP and Pi levels are restored in the small fibers
compared to the large fibers during recovery, as well as the relative
stability in ATP and total high-energy phosphate content during contraction
and recovery in both size classes. The arrow indicates when burst contractile
exercise was stimulated; the asterisk indicates that values are significantly
different from the resting value; and the dagger indicates that AP levels in
each size class were significantly different from each other at the common 15
min recovery time point. N≥7 for every point.

Relative changes in glycogen content in small (open symbols) and large
(filled symbols) dark levator fibers during a contraction-recovery cycle.
Values at each time point have been normalized to the mean resting value to
allow direct comparison between the size classes (absolute resting values are
in Table 3). The arrow
indicates when burst contractile exercise was stimulated. The asterisk
indicates values significantly different from the resting value.
N≥10 for every point.

Post-contractile AP resynthesis in anaerobic light fibers from both
juvenile and adult blue crabs has been shown to occur in about 60 min, despite
large differences in body mass and fiber size
(Kinsey et al., 2005). This
size independence appears to result from anaerobic contributions to recovery
in large fibers (Johnson et al.,
2004). Post-contractile AP resynthesis in the aerobic fibers,
however, should be much faster owing to a nearly tenfold greater mitochondrial
content and a twofold greater citrate synthase (CS) activity than light fibers
(Boyle et al., 2003;
Johnson et al., 2004). This is
supported by our observation that AP recovery in the small dark fibers was
complete 15 min after exercise, a rate of recovery roughly four times faster
than found in the light fibers. However, the dark fibers of adults recovered
at the same rate as previously observed for the light fibers. Further, the
relatively fast AP recovery rate in the small dark fibers was still several
fold lower than typical recovery rates in mammalian skeletal muscle with a
similar mitochondrial content (e.g. Meyer,
1988; Vicini and Kushmerick,
2000; Hancock et al.,
2005). Thus, the AP recovery rate appears to be influenced by both
size-dependent and size-independent factors.

Measured AP recovery (symbols) compared to the volume averaged model of AP
recovery (solid line) in small (A) and large (B) dark fibers. The dotted line
indicates the resting concentration. In the model, the myosin ATPase was
activated long enough to cause a decrease in AP that was comparable to the
measured data. (C,D) Three-dimensional graphs show the temporally and
spatially resolved concentrations of AP for small (C) and large (D) dark
levator fibers during a contraction-recovery cycle. This model output was
generated using parameters in Table
2. Note the absence of concentration gradients.

In the large dark fibers, intracellular subdivisions serve to create
metabolic functional units with the same small diameter as the juvenile
fibers, thereby permitting sustained aerobic contraction. Additionally,
mitochondria represent the same total fractional volume (23-25%) in both small
and large dark fiber subdivisions (Johnson
et al., 2004). Thus, we expected to see a rapid AP recovery in the
dark fibers from both size classes, with differences between size classes
attributable to body mass-specific metabolic scaling. The mass-specific
scaling exponent (b) for CS activity in blue crab aerobic fibers is
relatively small (b=-0.19)
(Johnson et al., 2004),
whereas in many mammalian systems measurements of basal rates of O2
consumption typically yield b values near -0.25
(Brody, 1945), although
b may be as high as -0.33 (White
and Seymour, 2003). Using this range of scaling exponents we
calculated that small dark fibers should recover two to four times faster than
large dark fibers due to scaling alone. In the present study, AP recovery in
the adults took around 60 min, which is roughly four times longer than in
juveniles. Whereas the observed size-dependence of AP recovery lies at the
upper range of that expected from mass-specific metabolic scaling, the low
scaling exponents for CS activity and mitochondrial density in blue crab
fibers suggests that scaling does not fully account for the difference in the
rate of AP recovery between juvenile and adult crabs.

What then can account for residual differences between size classes, and
why do the aerobic fibers recover more slowly than expected? The resynthesis
of phosphagens following contraction is a proton producing process. It has
been shown in vertebrate systems that changes in intracellular pH
(pHi) are responsible for altering the creatine kinase equilibrium
constant and hence, the phosphocreatine recovery rate
(Sahlin et al., 1975;
Harris et al., 1976;
Meyer et al., 1986;
van den Thillart and Waarde,
1993; McMahon and Jenkins,
2002). Similarly, experimental reductions in pHi in
invertebrate muscle lead to a reduction in AP concentration
(Combs and Ellington, 1995).
Considering that our exercise protocol leads to substantial lactate production
in all size classes (Johnson et al.,
2004), and since pH recovers with a time course similar to AP in
blue crab dark muscle (Milligan et al.,
1989), it is possible that a reduced pHi in the dark
fibers induces a transient shift in the AK equilibrium constant and slows the
rate of AP recovery in both small and large fibers. However, it is unlikely
that cellular acidosis can explain the differences in the rate of AP recovery
between the small and large fibers, since in adult animals the dark levator
pHi recovers to resting levels much faster than extracellular pH or
lactate concentration, despite the fact that anaerobic metabolism continues
after contraction (Milligan et al.,
1989). However, the processing of accumulated lactate may
contribute to the differences between size classes. There is evidence that
gluconeogenesis occurs in swimming muscle of blue crabs, and since there is no
known designated site for lactate processing in crustaceans comparable to the
mammalian liver, there is no Cori cycle
(Milligan et al., 1989;
Lallier and Walsh, 1991;
Henry et al., 1994). Thus,
lactate diffusing into the dark fibers of adult crabs may be used as a
substrate for gluconeogenesis. Since gluconeogenesis and glycolysis are
reciprocally controlled, aerobic AP recovery may be slowed in the large fibers
because they are supplied with lactate, whereas the small fibers are not.

The effect of increasing the rate of mitochondrial ATP production and
myosin ATPase activity during steady-state contraction in small fibers on the
temporal and spatial profiles of AP (A,C,E) and ATP (B,D,F). Metabolite
concentrations during a typical steady-state contraction, where
Vm,mito=1.00×10-14 mmolμ
m-2 s-1 and Vm,myo=1 mmol
l-1 s-1 (A,B), during steady-state with a threefold
increase in Vm,mito and Vm,myo (C,D),
and during steady-state with a sevenfold increase in
Vm,mito and Vm,myo (E,F).

An unexpected finding of Johnson et al.
(Johnson et al., 2004) was
that there was significant post-contractile lactate accumulation in the dark
fibers of the adult crabs, since it was assumed that their subdivisions
alleviate the need for anaerobic contributions during recovery. They reasoned
that this accumulation was likely a consequence of the dark fiber's close
proximity to lactate-producing light fibers and net diffusive flux into the
dark fibers from the lactate-laden hemolymph, and not a result of
post-contractile anaerobic glycogenolysis occurring within the dark fibers.
This supposition is supported by our observation that large dark fibers have
no significant post-contractile glycogen depletion
(Fig. 4). This is in contrast
to large light fibers, which produce copious lactate and significantly deplete
glycogen post-contraction (Boyle et al.,
2003; Johnson et al.,
2004).

Though the large fibers did not exhibit any significant post-contractile
glycogen depletion, we did observe a sharp, yet non-significant decrease in
glycogen immediately after exercise. This depletion may result from anaerobic
glycogenolysis, which increasingly powers burst contractions in these fibers
as AP is depleted, although a similar contraction-induced decrease was not
observed in small fibers despite producing an identical amount of lactate.
This same pattern of post-contractile glycogen depletion was observed by Henry
et al. (Henry et al., 1994) in
adult blue crab dark fibers following vigorous exercise, indicating that this
may be a typical response to burst contractile activity. Although we did not
observe any significant post-contractile glycogen depletion in the large
fibers as expected, we did see an unexpected depletion of glycogen during
recovery in the small fibers. Boyle et al.
(Boyle et al., 2003), who
measured post-contractile glycogen dynamics in blue crab light fibers,
reported no significant glycogen depletion during recovery in the juveniles.
However, immediately after exercise and before sacrifice the animals in their
study were fed, potentially restoring depleted glycogen pools during the
several hours of recovery. In our study, animals were not provided with a food
source during recovery. We speculate that with no glycogen storing organ
(van Aardt, 1988;
Lallier and Walsh, 1991;
Henry et al., 1994), as in the
mammalian liver, and with no new source of glucose from a food supply,
glycogen pools diminished by aerobic glycogenolysis during recovery could not
be replenished to resting levels.

Kinsey et al. (Kinsey et al.,
2005) used the same mathematical reaction-diffusion model used in
the present study to investigate whether diffusion was limiting to AP recovery
in the blue crab anaerobic light muscle; a fiber with extreme proportions, but
a relatively low aerobic capacity (and hence low rate of ATP production). They
found only small intracellular concentration gradients of high-energy
phosphates during simulations of AP recovery. However, gradients became more
substantial as the mitochondrial reaction rate was increased, which
illustrated the interaction between diffusion limitation and ATP turnover
rates. Intracellular diffusive flux does not appear to exert substantial
control over AP recovery in the blue crab giant light fibers, but there may be
other cell types where diffusion is limiting. These are likely to include
systems with a relatively high rate of ATP production/consumption such as the
blue crab dark fibers, which have a 10-fold higher mitochondrial density than
the light fibers. However, our reaction-diffusion analysis revealed no
intracellular concentration gradients of high-energy phosphates during a burst
contraction-recovery cycle at the rates of AP recovery determined for the dark
fibers (Fig. 5). This is not
surprising considering that AP recovery rates in dark fibers were similar to
those found previously for light fibers
(Kinsey et al., 2005),
although it is likely that a less intense exercise protocol may have allowed
higher recovery rates in dark fibers by reducing lactate production (see
above). In fact, in prior simulations where the mitochondrial rate was
increased to yield AP recovery rates that were comparable to that observed in
aerobic mammalian muscle, substantial gradients were observed
(Kinsey et al., 2005).
Nevertheless, even the fast rate of recovery that was observed in the dark
fibers of the small animals was too slow to be limited by intracellular
metabolite diffusion, which is consistent with the analysis of light fibers by
Kinsey et al. (Kinsey et al.,
2005).

Although post-contractile AP recovery in the dark fibers does not appear to
be limited by diffusion, these aerobic fibers normally power steady-state
contraction during sustained swimming. Under these conditions, ATP turnover
rates are much higher than they are during post-contractile recovery. Using a
myosin ATPase rate that was 25% of that used for burst contraction and a
Vm,mito that is comparable to that of prior studies (see
Vicini and Kushmerick, 2000),
we observed substantial concentration gradients in AP and ATP, as well as
other metabolites, during simulated steady-state contraction. As expected, the
gradients became more substantial as the ATP turnover rate was increased
(Fig. 6). The ATP buffering
role of AK is apparent since at relatively low rates of demand ATP gradients
are minimal, but as AP is depleted ATP gradients become severe. Thus, it
appears that AP recovery in blue crab dark fibers is not limited by diffusion
at the low rates of ATP turnover that seem to characterize our burst
contraction-recovery protocol, but despite the short intracellular diffusion
distances resulting from fiber subdivisions, the high rates of ATP turnover
observed during steady-state contraction result in substantial metabolite
gradients.

In summary, the patterns of recovery that have been observed in blue crab
locomotor muscles previously (Boyle et al.,
2003; Johnson et al.,
2004; Kinsey et al.,
2005), and herein, suggest that there are effects of fiber size on
aerobic metabolism. Although the aerobic dark fibers are as large as the
anaerobic light fibers, the selective pressure to power aerobic swimming has
promoted the evolution of an intricate network of mitochondria-rich, highly
perfused subdivisions. These subdivisions allow the fibers to retain a small
metabolic functional unit while apparently developing a large contractile
functional unit during growth, thereby eliminating the need for anaerobic
contributions to recovery in adult animals. Our reaction-diffusion analysis,
in conjunction with observed AP recovery data, suggests that intracellular
diffusion does not limit aerobic recovery in blue crab levator fibers, as
expected. However, the rates of AP recovery in the dark fibers were
considerably lower than expected, considering the high mitochondrial density
of these fibers, and this was probably due to metabolic inhibition. During
simulated steady-state contraction, intracellular metabolite diffusion did
limit aerobic metabolism, suggesting that diffusion may exert substantial
control over aerobic flux even in small fibers if the ATP turnover rate is
high.

ACKNOWLEDGEMENTS

This research was supported by National Science Foundation grants to S.T.K.
(IBN-0316909) and B.R.L. (IBN-0315883), as well as a Sigma Xi Grant-in-Aid of
Research to K.M.H.

Other journals from The Company of Biologists

Our most recent Editors’ choice is a pair of articles that examine rapid depth perception in hunting archerfish from Caroline P. Reinel and Stefan Schuster (I. The predictive C-starts, II. An analysis of potential cues). Archerfish have an impressive ability to predict where the prey that they topple from perches will land and now it turns out that they are able to calculate the height that a victim will fall in the first 100 ms of the descent.

Animals that live in the air have high levels of CO2 in their bodies, while aquatic species have lower CO2 levels, but what about the internal CO2 levels of animals that evolved in air but returned to the water for part of their lives? Philip Matthews and colleagues have discovered that the CO2 in the haemolymph of aquatic dragonfly nymphs is high, like that of air breathers, even though they live in water.

“So much of science is about things going wrong, failing epically, and I think you have to be able to pick yourself up and figure out what went wrong.”

Erika Eliason is an Assistant Professor at University of California, Santa Barbara, USA, where she studies ecological and evolutionary physiology. She shares how a childhood spent discovering the natural world in the woods where she grew up ignited her love for science and fieldwork, why conferences should be more family-friendly and the importance of being prepared for the unexpected.

Exposure to low temperature often results in death or injury for insects. However, repeated brief warm pulses during periods of low-temperature stress have been shown to confer some protection. Kendra J. Greenlee and colleagues provide an overview of the critical elements that underpin this phenomenon.

Meet the team

Are you going to the 2018 APS Comparative Physiology meeting in October? This meeting is supported by JEB and our Reviews Editor Charlotte Rutledge will be there - stop by and say hello! Advance registration is open until 24 September 2018.